Human chorionic gonadotropin-dependent induction of an equine aldo-keto reductase (AKR1C23) with 20α-hydroxysteroid dehydrogenase activity during follicular luteinization in vivo

Aldo–keto reductases (AKRs) are multifunctional enzymes capable of acting on a wide variety of substrates, including sex steroids. AKRs having 20α-hydroxysteroid dehydrogenase (20α-HSD) activity can reduce progesterone to 20α-hydroxy-4-pregnen-3-one (20α-DHP), a metabolite with lower affinity for the progesterone receptor. The objective of this study was to investigate the regulation of equine AKR1C23 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The equine AKR1C23 cDNA was cloned and shown to encode a 322 amino acid protein that is conserved (71–81% identity) when compared with mammalian orthologs. RT-PCR/Southern blotting analyses were performed to study the regulation of AKR1C23 transcripts in equine preovulatory follicles isolated between 0 and 39 h after hCG treatment (ovulation occurring 39–42 h post-hCG). Results showed the presence of low AKR1C23 expression before hCG treatment, but a marked increase was observed in follicles obtained 12 h after hCG (P<0.05). Analyses of isolated preparations of granulosa and theca interna cells identified low mRNA expression in both cell types prior to hCG treatment, with granulosa cells clearly being the predominant site of follicular AKR1C23 mRNA induction. A specific polyclonal antibody was raised against a fragment of the equine protein and immunoblotting analyses showed an increase in AKR1C23 protein in granulosa cell extracts when comparing follicles isolated at 36 h post-hCG vs those collected prior to treatment, in keeping with mRNA results. Immunohistochemical data confirmed the induction of the enzyme in follicular cells after hCG treatment. The enzyme was tested for 20α-HSD activity and was shown to exhibit a KM of 3.12 μM, and a Vmax of 0.86 pmol/min per 10 μg protein towards progesterone. The levels of 20α-DHP measured in follicular fluid reflected this activity. Collectively, these results demonstrate for the first time that the gonadotropin-dependent induction of follicular luteinization is accompanied by an increase in AKR1C23 expression. Considering the 20α-HSD activity of AKR1C23, its regulated expression in luteinizing preovulatory follicles may provide a biochemical basis for the increase in ovarian 20α-DHP observed during gonadotropin-induced luteinization/ovulation. (The nucleotide sequence reported in this paper has been submitted to GenBank with accession number AY955082.)

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Human Chorionic Gonadotropin-Dependent Regulation of 17β-Hydroxysteroid Dehydrogenase Type 4 in Preovulatory Follicles and Its Potential Role in Follicular Luteinization

Endocrinology ◽

10.1210/en.2003-1715 ◽

2004 ◽

Vol 145 (4) ◽

pp. 1906-1915 ◽

Cited By ~ 17

Author(s):

Kristy A. Brown ◽

Derek Boerboom ◽

Nadine Bouchard ◽

Monique Doré ◽

Jacques G. Lussier ◽

...

Keyword(s):

Mrna Expression ◽

Chorionic Gonadotropin ◽

Human Chorionic Gonadotropin ◽

Hydroxysteroid Dehydrogenase ◽

Mrna Induction ◽

Multidomain Structure ◽

Acid Protein ◽

Preovulatory Follicles ◽

17Β Estradiol ◽

Theca Interna

Abstract 17β-Hydroxysteroid dehydrogenase type 4 (17βHSD4) has a unique multidomain structure, with one domain involved in 17β-estradiol inactivation. The objective of the study was to investigate the regulation of 17βHSD4 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The equine 17βHSD4 cDNA was cloned and was shown to encode a 735-amino acid protein that is highly conserved (81–87% identity) compared with other mammalian orthologs. RT-PCR/Southern blot analyses were performed to study the regulation of 17βHSD4 transcripts in equine preovulatory follicles isolated between 0–39 h after hCG treatment. Results showed the presence of basal 17βHSD4 mRNA expression before hCG treatment, but an increase was observed in follicles obtained 24 h after hCG (P < 0.05). Analyses of isolated preparations of granulosa and theca interna cells identified basal mRNA expression in both layers, but granulosa cells appeared as the predominant site of follicular 17βHSD4 mRNA induction. A specific polyclonal antibody was raised against a fragment of the equine protein and used to study regulation of the 17βHSD4 protein. Immunoblots showed an increase in full-length 17βHSD4 protein in follicles 24 h after hCG (P < 0.05), in keeping with mRNA results. Immunohistochemical data confirmed the induction of the enzyme in follicular cells after hCG treatment. Collectively, these results demonstrate that the gonadotropin-dependent induction of follicular luteinization is accompanied by an increase in 17βHSD4 expression. Considering the estrogen-inactivating function of 17βHSD4, its regulated expression in luteinizing preovulatory follicles appears as a potential complementary mechanism to reduce circulating levels of 17β-estradiol after the LH surge.

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Human Chorionic Gonadotropin-Dependent Up-Regulation of Genes Responsible for Estrogen Sulfoconjugation and Export in Granulosa Cells of Luteinizing Preovulatory Follicles

Endocrinology ◽

10.1210/en.2006-0420 ◽

2006 ◽

Vol 147 (9) ◽

pp. 4222-4233 ◽

Cited By ~ 9

Author(s):

Kristy A. Brown ◽

Monique Doré ◽

Jacques G. Lussier ◽

Jean Sirois

Keyword(s):

Chorionic Gonadotropin ◽

Granulosa Cells ◽

Human Chorionic Gonadotropin ◽

Cell Layer ◽

Rt Pcr ◽

Expression Of Genes ◽

Preovulatory Follicles ◽

Steady State Levels ◽

17Β Estradiol ◽

Theca Interna

Estrogen sulfotransferase (EST) is responsible for the sulfoconjugation of estrogens, thereby changing their physical properties and preventing their action via the estrogen receptors. These sulfoconjugated steroids no longer diffuse freely across the lipid bilayer; instead, they are exported by members of the ATP-binding cassette family, such as ABCC1. The objective of this study was to investigate the regulation of EST and ABCC1 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The transcripts for EST and ABCC1 were cloned by RT-PCR, and the regulation of their mRNAs was studied in preovulatory follicles obtained during estrus at 0, 12, 24, 30, 33, 36, and 39 h after hCG. Results obtained from RT-PCR/Southern blot analyses showed significant changes in steady-state levels of both EST and ABCC1 mRNA after hCG treatment (P < 0.05). In granulosa cells, a significant increase in EST transcript was observed 30–39 h after hCG. Similarly, ABCC1 transcript levels were induced in granulosa cells 12–39 h after hCG. In contrast, no significant changes in either EST or ABCC1 were detected in theca interna samples after hCG. The increase in EST and ABCC1 transcripts observed in granulosa cells was reflected in preparations of intact follicle walls, suggesting that the granulosa cell layer contributes the majority of EST and ABCC1 expression in preovulatory follicles. The present study demonstrates that follicular luteinization is accompanied not only by a decrease in 17β-estradiol biosynthesis but also by an increase in expression of genes responsible for estrogen inactivation and elimination from granulosa cells, such as EST and ABCC1, respectively.

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Effects of the antiandrogen hydroxyflutamide on progesterone secretion by preovulatory rat follicles in vivo and in vitro

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y91-189 ◽

1991 ◽

Vol 69 (9) ◽

pp. 1288-1293 ◽

Cited By ~ 1

Author(s):

Yallampalli Chandrasekhar ◽

David T. Armstrong

Keyword(s):

Luteinizing Hormone ◽

Chorionic Gonadotropin ◽

Human Chorionic Gonadotropin ◽

Serum Progesterone ◽

Antagonistic Action ◽

Lh Surge ◽

Progesterone Secretion ◽

Preovulatory Follicles

Serum and ovarian progesterone levels and in vitro production of progesterone by preovulatory follicles were measured on proestrus in pregnant mare's serum gonadotropin (PMSG) primed immature rats in which the luteinizing hormone (LH) surge and ovulation were blocked by administration of the antiandrogen hydroxyflutamide. Serum progesterone levels observed at 12:00 on proestrus were significantly elevated, twofold above those observed in vehicle-treated controls, by in vivo administration of 5 mg hydroxyflutamide 4 h earlier. In control rats, proestrous progesterone did not increase until 16:00, in parallel with rising LH levels of the LH surge. No LH surge occurred in the hydroxyflutamide-treated rats, ovulation was blocked, and serum progesterone declined throughout the afternoon of proestrus, from the elevated levels present at 12:00. Administration of human chorionic gonadotropin (hCG) at 11:00 advanced the elevation of serum progesterone by 2 h in vehicle-treated controls and prevented the decline in progesterone levels in hydroxyfiutamide-treated rats. The patterns of change in ovarian tissue concentrations with time and treatment were essentially similar to those observed for serum progesterone. In in vitro experiments, progesterone secretion during 24 h culture of preovulatory follicles obtained on PMSG-induced proestrus was significantly increased, sixfold, by addition to the culture media of 370 μM but not of 37 μM hydroxyflutamide. Testosterone (50 nM) and hCG (20 mIU/mL) caused 26- and 14-fold increases, respectively, in progesterone secretion by cultured follicles. Hydroxyflutamide significantly reduced the stimulatory effect of testosterone but not of hCG on progesterone secretion in vitro. These results suggest that the antiandrogen hydroxyflutamide stimulates progesterone secretion, both in vivo and in vitro, through an initial androgen-agonistic action, before its antagonistic action is expressed. Its androgen-antagonistic action is responsible for its ability to inhibit testosterone-induced progesterone secretion in vitro. Its failure to reduce hCG-stimulated progesterone secretion in vivo and in vitro indicates that the latter stimulation is exerted independently of, and not as a consequence of, androgen action. The decrease in serum progesterone levels on the afternoon of proestrus therefore appears to be a consequence rather than a cause of the absence of an LH surge in the hydroxyflutamide-treated rats. It is concluded that the inhibitory effect of hydroxyflutamide on the preovulatory LH surge and ovulation is due not to inhibition of progesterone secretion at the ovarian level but most likely to neuroendocrine site(s) of action of the inhibitor.Key words: antiandrogen, hydroxyflutamide, progesterone, luteinizing hormone, ovulation, human chorionic gonadotropin.

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Regulation of transcripts encoding ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs-1) and progesterone receptor by human chorionic gonadotropin in equine preovulatory follicles

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0310473 ◽

2003 ◽

Vol 31 (3) ◽

pp. 473-485 ◽

Cited By ~ 33

Author(s):

D Boerboom ◽

DL Russell ◽

JS Richards ◽

J Sirois

Keyword(s):

Progesterone Receptor ◽

Chorionic Gonadotropin ◽

Granulosa Cells ◽

Human Chorionic Gonadotropin ◽

Reproductive Tract ◽

Pcr Analysis ◽

Preovulatory Follicles ◽

Theca Interna ◽

A Disintegrin And Metalloproteinase ◽

Ovulatory Process

One member of a new family of metalloproteinases, a disintegrin and metalloproteinase with thrombospondin-like motifs-1 (ADAMTS-1), has been found to be expressed and hormonally induced in granulosa cells of ovulating rodent follicles. Furthermore, the targeted disruption of the ADAMTS-1 gene resulted in ovarian defects associated with severely impaired fertility. While these data demonstrate the importance of ADAMTS-1 in rodent ovarian physiology, the potential role of ADAMTS-1 in the ovulatory process of monoovulatory species remains unknown. The objectives of this study were to clone the equine ADAMTS-1 primary transcript and to study its regulation during human chorionic gonadotropin (hCG)-induced ovulation. A 3573 bp follicular cDNA library clone was isolated and found to encode a nearly complete, highly conserved ADAMTS-1 homologue. Real-time RT-PCR analysis detected this transcript in diverse tIssues, including previously unreported sites of ADAMTS-1 expression such as the male reproductive tract, the follicular theca interna and the mature corpus luteum. The tIssue distribution of the progesterone receptor (PR), a known regulator of ADAMTS-1 expression in rodent preovulatory follicles, was found to overlap that of ADAMTS-1 in some tIssues. A study of the regulation of follicular ADAMTS-1 and PR mRNAs during the hCG-induced ovulatory process revealed distinct patterns of regulation in granulosa cells and in theca interna. In granulosa cells, ADAMTS-1 mRNA was found to be induced at 12 h post-hCG (P<0.05), followed by a return to basal levels by 30 h and a re-increase at 33-39 h (P<0.05). A concomitant increase in PR mRNA (P<0.05) was observed at 12 h post-hCG. In theca interna, abundant ADAMTS-1 mRNA was detected at all timepoints, and levels increased transiently at 33 h post-hCG (P<0.05), whereas no significant change was observed in PR mRNA. Together, these data demonstrate for the first time the hormonally regulated ovarian expression of ADAMTS-1 in a monoovulatory species, and identify a novel biphasic regulation of ADAMTS-1 in granulosa cells and a regulated expression in theca interna that were not previously observed in rodents.

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